The availability of insulin replacement therapy has prevented the mortality and morbidity of acute complications in diabetes mellitus. However, chronic diabetic complications remain a major health problem due to persistent metabolic derangement, arising principally from poor control of blood glucose. Results emerging from the Diabetes Control and Complications Trial (DCCT) indicate that a decrease of 1% in Hb Alc correlates with more than 35% improvement in the incidence of retinopathy.
In order to achieve normal glycemia, therapy must be designed to parallel as closely as possible the pattern of endogenous insulin secretion in normal individuals. The daily physiological demand for insulin fluctuates and can be separated into two phases: (a) the absorptive phase requiring a pulse of insulin to dispose of the meal-related blood glucose surge, and (b) the post-absorptive phase requiring a sustained amount of insulin to regulate hepatic glucose output for maintaining optimal fasting blood glucose. Accordingly, effective therapy involves the combined use of two types of exogenous insulin: a fast-acting meal time insulin and a long-acting basal insulin.
To achieve a long-acting basal time action, insulin is currently formulated under conditions favoring formation of a hexamer conformation in an insoluble, crystalline state. These long acting formulations are Ultralente, Lente, and semi-Lente. However, the insolubility of the current long-acting preparations has been shown to cause problems relating to inconsistency in the dose-response as well as unpredictability in time action. In addition, one of the currently available long-acting insulin preparations, beef Ultralente, is immunogenic. The presence of antibodies that results from the immunogenicity of beef Ultralente alters the pharmacokinetics of fast-acting insulins.
While the time action of the insoluble Ultralente formulation makes a convenient once-a-day basal insulin, many physicians actually prefer to use an intermediate time action insulin, an insulin-protamine formulation commonly referred to as insulin-NPH. Insulin-NPH is used twice daily as a basal insulin because it is comparatively easier to adjust the optimal dosage with a drug of shorter time action. As a result, intermediate-acting insulins account for 70% of the US, 64% of the Japanese, 45% of European and an overall 55% of the world-wide insulin market.
However, both insoluble insulin-NPH and insoluble Ultralente insulin are suspension formulations. Thus, the formulations are inherently less predictable than soluble formulations and result in less than adequate control of blood glucose and a greater susceptibility to life-threatening hypoglycemic episodes. Accordingly, there remains a need for a soluble, long-acting basal insulin in order to achieve successful intensive insulin replacement therapy. The present invention provides acylated insulin analogs that may be formulated to provide soluble, basal insulin therapy.
The acylation of pork, beef, or human insulin is disclosed by Muranishi and Kiso, in Japanese Patent Application 1-254,699. The following compounds are specifically disclosed: B29-N.sup..epsilon. -palmitoyl insulin (the .epsilon.-amino group is acylated), B1-N.sup..alpha. -palmitoyl insulin (the N terminal .alpha.-amino group of the B chain is acylated), and B1,B29-N.sup..alpha.,N.sup..epsilon. -dipalmitoyl insulin (both the .epsilon.-amino and the N-terminal .alpha.-amino group are acylated). Muranishi and Kiso disclose that acylated insulin possesses a biological profile similar to insulin; but fails to provide the dosages, routes of administration, or other conditions of the in vivo model to allow one skilled in the art to evaluate the activity or duration of action of the acylated insulin.
Similarly, Hashimoto et al., in Pharmaceutical Research 6: 171-176 (1989), disclose B1-N.sup..alpha. -palmitoyl insulin (the N terminal .alpha.-amino group is acylated), and B1,B29-N.sup..alpha.,N.sup..epsilon. -dipalmitoyl insulin (both the .epsilon.-amino and the N-terminal .alpha.-amino groups are acylated). Hashimoto et al. studied the hypoglycemic effect of B1-N.sup..alpha. -palmitoyl insulin and B1,B29-N.sup..alpha., N.sup..epsilon. -dipalmitoyl insulin in male rats at 25 U/mL, an exceedingly high dose. At these doses, FIG. 5 demonstrates very low activity when administered intravenously. When administered intramuscularly, only a short hypoglycemic effect of B1-N.sup..alpha. -palmitoyl insulin and negligible effect of B1,B29-N.sup..alpha.,N.sup..epsilon. -dipalmitoyl insulin were disclosed in FIG. 6.
In addition to the in vivo reports by Muranishi and Kiso and Hashimoto et al., Walder et al., in PCT publication WO 92/01476, disclose that the half-life of proteins and peptides can be extended in vivo by chemically linking the protein with an apolar group, specifically a fatty acid derivative. The fatty acid provides a bridging group between the protein and albumin. Walder et al. continue to disclose that the apolar group is preferably restricted to a unique site or sites in the protein and exemplify the binding of the cysteine residues of hemoglobin. The reference generally discloses fatty acid derivatives of insulin. However, no fatty acid derivatives of insulin are specifically disclosed or exemplified, and no data are disclosed to indicate that the biological activity of the fatty acid derivatives of insulin is retained.
It has been discovered that the selective acylation of a free amino group of a monomeric insulin analog provides effective basal insulin activity. The unacylated insulin analogs described herein are designed to provide a rapid onset of action and a rapid clearance. These analogs are known in the art as monomeric insulin analogs. The ability to modify such analogs to provide basal activity is most unexpected.
The present invention provides a mono-acylated insulin analog that yields upon use an extended duration of action. The analogs may be prepared in soluble formulations thus offering advantages over current basal insulin therapy. The present analogs also possess excellent predictability in dose response, excellent predictability in time action, lack a distinct peak in the time-action profile, and are ideally suited for the preparation of mixture formulations comprising an insulin analog and acylated insulin analog.